Altered leukotriene B4 metabolism in colonic mucosa with inflammatory bowel disease.

BACKGROUND: omega-Oxidation is regarded as the major pathway for leukotriene B4 (LTB4) metabolism. Very little is known about it in colonic mucosa with inflammatory bowel disease (IBD). METHODS: We investigated the metabolic ratio of omega-oxidation to LTB4 biosynthesis in colonic mucosa from patients with IBD and control subjects. After incubation of colonic mucosa with 14C-arachidonic acid and ionophore A23187, we separated LTB4 and its omega-oxidative metabolites by high-performance liquid chromatography. RESULTS: The rate of LTB4 omega-oxidation was comparable to the rate of its biosynthesis. The metabolic ratio was significantly decreased in inflamed mucosa with ulcerative colitis. CONCLUSIONS: LTB4 omega-hydroxylase activity is an important factor in regulating LTB4 level in colonic mucosa, and the increased LTB4 level in inflamed mucosa with IBD, especially ulcerative colitis, is caused by decreased LTB4 omega-hydroxylase activity and increased 5-lipoxygenase activity.

5-Aminosalicylic acid inhibits leukotriene B4 omega-hydroxylase activity in human polymorphonuclear leukocytes.

omega-Oxidation is regarded as the major pathway for the metabolism and inactivation of leukotriene B4 (LTB4). To investigate the action of 5-aminosalicylic acid (5-ASA) on LTB4 omega-hydroxylase activity, we incubated human polymorphonuclear leukocytes (PMNLs) with 3H-labeled LTB4 after pre-incubation with various concentrations of 5-ASA. omega-oxidation metabolites were separated by high performance liquid chromatography and each radioactivity was measured by a liquid scintilation counter. LTB4 omega-hydroxylase activity was inhibited by 5-ASA in a concentration-dependent fashion. The 50% inhibitory dose was about 50 mumol/l, which is within the concentration range found in the colonic mucosa. Our findings may be valuable in elucidating the potentially critical aspect of 5-ASA treatment in ulcerative colitis (UC).

Leukotriene B4 omega-hydroxylase activity in polymorphonuclear leukocytes from patients with inflammatory bowel disease.

omega-oxidation is regarded as the major pathway for the catabolism of leukotriene B4 (LTB4). To determine how LTB4 omega-hydroxylase is modulated in inflammatory bowel disease, we investigated the kinetic characteristics of this enzyme in 10 patients with Crohn's disease (CD), nine with ulcerative colitis (UC) and eight healthy control subjects. After incubating polymorphonuclear leukocytes with various concentrations of 3H-labeled LTB4, omega-oxidation products were separated by high performance liquid chromatography (HPLC) and the radioactivity was measured by a liquid scintillation counter. The apparent Vmax values were significantly higher in both disease than in healthy control subjects, although the difference between CD and UC was insignificant. There was no difference in the apparent Km values. And the Vmax/Km ratios of patients with CD were significantly higher than that of healthy control subjects. It is suggested that LTB4 metabolism is activated in inflammatory bowel disease (IBD) and that the modulation of this enzyme activity has an important role in the pathogenesis of inflammatory bowel disease.

Effect of intravenously infused eicosapentaenoic acid on the leukotriene generation in patients with active Crohn's disease.

Ten patients with active Crohn's disease who have been managed with parenteral-nutrition therapy were administered a lipid emulsion either with [containing 0.6 g eicosapentaenoic acid (EPA)] or without fish oil for 2 wk. We isolated polymorphonuclear leukocytes (PMNLs) from the patients before and after this treatment and measured the amounts of leukotriene B4 (LTB4) and leukotriene B5 (LTB5) generated by activated PMNLs by reversed-phase HPLC. The LTB5 generation in active Crohn's disease before this treatment was significantly lower than in healthy control subjects. The amount of LTB5 and the LTB5-LTB4 ratio increased significantly after fish-oil supplementation. The difference with LTB4 was not statistically significant. We have shown that daily intravenous administration of 0.6 g EPA influenced the generation of leukotrienes in active Crohn's disease even after short-term treatment. Further investigations are necessary to clarify the correlation between EPA and clinical improvement in Crohn's disease.

Degradation of nucleic acids with ozone. II. Degradation of yeast RNA, yeast phenylalanine tRNA and tobacco mosaic virus RNA.

The degradation of a mixture of four 5'-ribonucleotides (AMP, GMP, CMP and UMP), yeast RNA, yeast phenylalanine tRNA, and tobacco mosaic virus RNA (TMV-RNA) with ozone (concentration in inlet gas, 0.1-0.5 mg/l) was examined in a phosphate buffer (pH 6.9). In the case of the mixture, GMP alone was degraded in the initial stage. In the ozonization of yeast RNA, the guanine moiety was less vulnerable to attack by ozone than in the case of free GMP, but it again degraded most rapidly among the four nucleotides. In the treatment of tRNA with ozone, the guanine moiety degraded first. When the numbers of degraded nucleotides reached 4.8 (remaining amino acid acceptor activity was 3.6%), the polyacrylamide gel electrophoresis of the ozonized tRNA gave a single band with the same mobility as that of the intact tRNA. It is evident that ozonolysis of tRNA proceeded without cleavage of the polynucleotide chain. In the case of TMV-RNA, the loss of the infectivity by ozone proceeded rapidly within 30 min and was followed by preferential degradation of the guanine moiety. The outstanding lability of the guanine moiety observed in each case is discussed in connection with the inactivation of tRNA and TMV-RNA.

Mode of degradation of tRNAs with ozone.

Ozone-treatment of tRNA resulted in the degradation of guanine residues located on the loop regions, such as the anticodon and D-loop region. In addition, it became evident that the guanine residues in the consecutive sequences, such as G-G-G-G and G-G-m1G in tRNAPro, were the most susceptive to the ozone-treatment. The internucleotidic linkage of the treated tRNA was not cleaved but several fragments were obtained by a gel electrophoretic separation after heating at 60% in 1M aniline-acetate buffer (pH 4.5). Major fragments derived from tRNAPro were the 3'-half and the 5'-half molecules produced by the cleavage at the anticodon region.